Immunomodulatory oligonucleotides

ABSTRACT

Oligonucleotides containing unthylated CpG dinucleotides and therapeutic utilities based on their ability to stimulate an immune response in a subject are disclosed. Also disclosed are therapies for treating diseases associated with immune system activation that are initiated by unthylated CpG dinucleotides in a subject comprising administering to the subject oligonucleotides that do not contain unmethylated CpG sequences (i.e. methylated CpG sequences or no CpG sequence) to outcompete unmethylated CpG nucleic acids for binding. Further disclosed are methylated CpG containing dinucleotides for use antisense therapies or as in vivo hybridization probes, and immunoinhibitory oligonucleotides for use as antiviral therapeutics.

GOVERNMENT SUPPORT

The work resulting in this invention was supported in part by NationalInstitute of Health Grant No. R29-AR42556-01. The U.S. Government maytherefore be entitled to certain rights in the invention.

BACKGROUND OF THE INVENTION

DNA Binds to Cell Membrane and is Internalized

In the 1970's, several investigators reported the binding of highmolecular weight DNA to cell membranes (Lerner, R. A., W. Meinke, and D.A. Goldstein. 1971. “Membrane-associated DNA in the cytoplasm of diploidhuman lymphocytes”. Proc. Natl. Acad. Sci. USA 68:1212; Agrawal, S. K,R. W. Wagner, P. K. McAllister, and B. Rosenberg.

1975. “Cell-surface-associated nucleic acid in tumorigenic cells madevisible with platinum-pyrimidine complexes by electron microscopy”.Proc. Nat. Acad. Sci. USA 72:928). In 1985 Bennett et al. presented thefirst evidence that DNA binding to lymphocytes is similar to a ligandreceptor interaction: binding is saturable, competitive, and leads toDNA endocytosis and degradation (Bennett, R. M., G. T. Gabor, and M. M.Merritt, 1985. “DNA binding to human leukocytes. Evidence for areceptor-mediated association, internalization, and degradation of DNA”.J Clin. Invest. 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) areable to enter cells in a saturable, sequence independent, andtemperature and energy dependent fashion (reviewed in Jaroszewski, J.W., and J. S. Cohen. 1991. “Cellular uptake of antisenseoligodeoxynucleotides”. Advanced Drug Delivery Reviews 6:235; Akhtar,S., Y. Shoji, and R. L. Juliano. 1992. “Pharmaceutical aspects of thebiological stability and membrane transport characteristics of antisenseoligonucleotides”. In: Gene Regulation: Biology of Antisense RNA andDNA. R. P. Erickson, and J. G. Izant, eds. Raven Press, Ltd. New York,pp. 133; and Zhao, Q., T. Waldschimdt, E. Fisher, C. J. Herrera, and A.M. Krieg., 1994. “Stage specific oligonucleotide uptake in murine bonemarrow B cell precursors”. Blood, 84:3660). No receptor for DNA or ODNuptake has yet been cloned, and it is not yet clear whether ODN bindingand cell uptake occurs through the same or a different mechanism fromthat of high molecular weight DNA.

Lymphocyte ODN uptake has been shown to be regulated by cell activation.Spleen cells stimulated with the B cell mitogen LPS had dramaticallyenhanced ODN uptake in the B cell population, while spleen cells treatedwith the T cell mitogen Con A showed enhanced ODN uptake by T but not Bcells (Krieg, A. M., F. Grnelig-Meyling, M. F. Gourley, W. J. Kisch, L.A. Chrisey, and A. D. Steinberg. 1991. “Uptake ofoligodeoxyribonucleotides by lymphoid cells is heterogeneous andinducible”. Antisense Research and Development 1:161).

Immune Effects of Nucleic Acids

Several polynucleotides have been extensively evaluated as biologicalresponse modifiers. Perhaps the best example is poly (I,C) which is apotent inducer of IFN production as well as a macrophage activator andinducer of NK activity (Talmadge, J. E., J. Adams, H. Phillips, M.Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffmann, R. H. Wiltrout,and M. A. Chirigos. 1985. “Immunomodulatory effects in mice ofpolyinosinic-polycytidylic acid complexed with poly-L:-lysine andcarboxymethylcellulose”. Cancer Res. 45:1058; Wiltrout, R. H., R. R.Salup, T. A. Twilley, and J. E. Talmadge. 1985. “Immunomodulation ofnatural killer activity by polyribonucleotides”. J. Biol. Resp. Mod4:512; Krown, S. E. 1986. “Interferons and interferon inducers in cancertreatment”. Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp,J. W. Smith II, PG. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W. G.Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P.Creekmore. 1992. “Polyinosinic-polycytidylic acid complexed withpoly-L-lysine and carboxymethylcellulose in combination with interleukin2 in patients with cancer: clinical and immunological effects”. Canc.Res. 52:3005). It appears that this murine NK activation may be duesolely to induction of IFN β secretion (Ishikawa, R., and C. A. Biron.1993. “IFN induction and associated changes in splenic leukocytedistribution”. J. Immunol. 150:3713). This activation was specific forthe ribose sugar since deoxyribose was ineffective. Its potent in vitroantitumor activity led to several clinical trials using poly (I,C)complexed with poly-L-lysine and carboxymethylcellulose (to reducedegradation by RNAse) (Talmadge, J. E., et al., 1985. cited supra;Wiltrout, R. H., et al., 1985. cited supra); Krown, S. E., 1986. citedsupra); and Ewel, C. H., et al. 1992. cited supra). Unfortunately, toxicside effects have thus far prevented poly (I,C) from becoming a usefultherapeutic agent.

Guanine ribonucleotides substituted at the C8 position with either abromine or a thiol group are B cell mitogens and may replace “B celldifferentiation factors” (Feldbush, T. L., and Z. K. Ballas. 1985.“Lymphokine-like activity of 8-mercaptoguanosine: induction of T and Bcell differentiation”. J. Immunol. 134:3204; and Goodman, M. G. 1986.“Mechanism of synergy between T cell signals and C8-substituted guaninenucleosides in humoral immunity: B lymphotropic cytokines induceresponsiveness to 8-mercaptoguanosine”. J. Immunol. 136:3335).8-mercaptoguanosine and 8-bromoguanosine also can substitute for thecytokine requirement for the generation of MHC restricted CT1 (Feldbush,T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E.Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. “Activationof murine natural killer cells and macrophages by 8-bromoguanosine”. J.Immunol. 140:3249), and synergize with IL-2 in inducing murine LAKgeneration (Thompson, R. A., and Z. K. Ballas. 1990.“Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as anIL-2-sparing agent in LAK generation”. J. Immunol. 145:3524). The NK andLAK augmenting activities of these C8-substituted guanosines appear tobe due to their induction of IFN (Thompson, R. A., et al. 1990. citedsupra). Recently, a 5′ triphosphorylated thymidine produced by amycobacterium was found to be mitogenic for a subset of human γδ T cells(Constant, P., F. Davodeau, M. -A. Peyrat, Y. Poquet, G. Puzo, M.Bonneville, and J. -J. Fournie. 1994. “Stimulation of human γε T cellsby nonpeptidic mycobacterial ligands” Science 264:267). This reportindicated the possibility that the immune system may have evolved waysto preferentially respond to microbial nucleic acids.

Several observations suggest that certain DNA structures may also havethe potential to activate lymphocytes. For example, Bell et al. reportedthat nucleosomal protein-DNA complexes (but not naked DNA) in spleencell supernatants caused B cell proliferation and immunoglobulinsecretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990.“Immunogenic DNA-related factors”. J. Clin. Invest. 85:1487). In othercases, naked DNA has been reported to have immune effects. For example,Messina et al. have recently reported that 260 to 800 bp fragments ofpoly (dG)•(dC) and poly (dG•dC) were mitogenic for B cells (Messina, J.P., G. S. Gilkeson, and D. S. Pisetsky. 1993. “The influence of DNAstructure on the in vitro stimulation of murine lymphocytes by naturaland synthetic polynucleotide antigens”. Cell. Immunol. 147:148).Tokunaga, et al. have reported that dGe dC induces γ-IFN and NK activity(Tokunaga, S. Yamamoto, and K Namba. 1988. “A synthetic single-strandedDNA, poly(dG,dC), induces interferon-α/β and -γ, augments natural killeractivity, and suppresses tumor growth” Jpn. J Cancer Res. 79:682). Asidefrom such artificial homopolymer sequences, Pisetsky et al. reportedthat pure mammalian DNA has no detectable immune effects, but that DNAfrom certain bacteria induces B cell activation and immunoglobulinsecretion (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1991.“Stimulation of in vitro murine lymphocyte proliferation by bacterialDNA”. J. Immunol. 147:1759). Assuming that these data did not resultfrom some unusual contaminant, these studies suggested that a particularstructure or other characteristic of bacterial DNA renders it capable oftriggering B cell activation. Investigations of mycobacterial DNAsequences have demonstrated that ODN which contain certain palindromesequences can activate NK cells (Yanamoto, S., T. Yamamoto, T. Kataoka,E. Kuramoto, O. Yano, and T. Tokunaga. 1992. “Unique palindromicsequences in synthetic oligonucleotides are required to induce INF andaugment INF-mediated natural killer activity”. J. Immunol. 148:4072;Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino, S. Yamamoto, T.Yamamoto, T. Kataoka, and T. Tokunaga 1992. “Oligonucleotide sequencesrequired for natural killer cell activation”. Jpn. J Cancer Res.83:1128).

Several phosphorothioate modified ODN have been reported to induce invitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu, and W. E.Paul. 1992. “An antisense oligonucleotide complementary to a sequence inIγ2b increases γ2b germline transcripts, stimulates B cell DNAsynthesis, and inhibits immunoglobulin secretion”. J. Exp. Med 175:597;Branda, R. F., A. L. Moore, L. Mathews, J. J. McCormack, and G. Zon.1993. “Immune stimulation by an antisense oligomer complementary to therev gene of HIV-1”. Biochem. Pharmacol. 45:2037; McIntyre, K. W., KLombard-Gillooly, J. R Perez, C. Kunsch, U. M. Sarmiento, J. D. Larigan,K. T. Landreth, and R Narayanarn 1993. “A sense phosphorothioateoligonucleotide directed to the initiation codon of transcription factorNF-κ β T65 causes sequence-specific immune stimulation”. Antisense Res.Develop. 3:309; and Pisetsky, D. S., and C. F. Reich 1993. “Stimulationof murine lymphocyte proliferation by a phosphorothioate oligonucleotidewith antisense activity for herpes simplex virus”. Life Sciences54:101). These reports do not suggest a common structural motif orsequence element in these ODN that might explain their effects.

The CREB/ATF Family of Transcription Factors and their Role inReplication

The cAMP response element binding protein (CREB) and activatingtranscription factor (ATF) or CREB/ATF family of transcription factorsis a ubiquitously expressed class of transcription factors of which 11members have so far been cloned (reviewed in de Groot, R. P., and P.Sassone-Corsi: “Hormonal control of gene expression: Multiplicity andversatility of cyclic adenosine 3′,5′-monophosphate-responsive nuclearregulators”. Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson:“Transcriptional regulation by CREB and its relatives”. Biochim Biophys.Acta 1174:221, 1993.). They all belong to the basic region/leucinezipper (bZip) class of proteins. All cells appear to express one or moreCREB/ATF proteins, but the members expressed and the regulation of mRNAsplicing appear to be tissue-specific. Differential splicing ofactivation domains can determine whether a particular CREB/ATF proteinwill be a transcriptional inhibitor or activator. Many CREB/ATF proteinsactivate viral transcription, but some splicing variants which lack theactivation domain are inhibitory. CREB/ATF proteins can bind DNA ashomo- or hetero- dimers through the cAMP response element, the CRE, theconsensus form of which is the unmethylated sequence TGACGTC (binding isabolished if the CpG is methylated) (Iguchi-Ariga, S. M. M., and W.Schaffner: “CpG methylation of the cAMP-responsive enhancer/promotersequence TGACGTCA abolishes specific factor binding as well astranscriptional activation”. Genes & Develop. 3:612, 1989.).

The transcriptional activity of the CRE is increased during B cellactivation (Xie, H. T. C. Chiles, and T. L. Rothstein: “Induction ofCREB activity via the surface Ig receptor of B cells”. J. Immunol.151:880, 1993.). CREB/ATF proteins appear to regulate the expression ofmultiple genes through the CRE including immunologically important genessuch as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W. R.Waterman, A. C. Webb, and P. E. Auron: “Transcription factors NF-IL6 andCREB recognize a common essential site in the human prointerleukin 1βgene”. Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Hernandez, D.Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: “Antisense DNAinhibition of tumor growth induced by c-Ha-ras oncogene in nude mice”.Cancer Res. 53:577, 1993), IFN-β (Du, W., and T. Maniatis: “An ATF/CREBbinding site protein is required for virus induction of the humaninterferon B gene”. Proc. Natl. Acad. Sci. USA 89:2150, 1992),TGF-β1(Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: “Binding ofAP-1/CREB proteins and of MDBP to contiguous sites downstream of thehuman TGF-B 1 gene”. Biochim. Biophys. Acta 1219:55, 1994.), TGF-β2,class II MHC (Cox, P. M., and C. R. Goding: “An ATF/CREB binding motifis required for aberrant constitutive expression of the MHC class II DRapromoter and activation by SV40 T-antigen”. Nucl. Acids Res. 20:4881,1992.), E-selectin, GM-CSF, CD-8α, the germline Igα constant regiongene, the TCR Vβ gene, and the proliferating cell nuclear antigen(Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M.B. Prystowsky: “Promoter activity of the proliferating-cell nuclearantigen gene is associated with inducible CRE-binding proteins ininterleukin 2-stimulated T lymphocytes”. Mol. Cell. Biol. 14:4233,1994.). In addition to activation through the cAMP pathway, CREB canalso mediate transcriptional responses to changes in intracellular Ca⁺⁺concentration (Sheng, M., G. McFadden, and M. E. Greenberg: “Membranedepolarization and calcium induce c-fos transcription viaphosphorylation of transcription factor CREB”. Neuron 4:571, 1990).

The role of protein-protein interactions in transcriptional activationby CREB/ATF proteins appears to be extremely important. Activation ofCREB through the cyclic AMP pathway requires protein kinase A (PKA),which phosphorylates CREB³⁴¹ on ser¹³³ and allows it to bind to arecently cloned protein, CBP (Kwok, R. P. S., J. R. Lundblad, J. C.Chrivia, J. P. Richards, H. P. Bachinger, R. G. Brennan, S. G. E.Roberts, M. R Green, and R. H. Goodman: “Nuclear protein CBP is acoactivator for the transcription factor CREB”. Nature 370:223, 1994;Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smea, M. Karin,J. Feramisco, and M. Montminy: “Activation of cAMP and mitogenresponsive genes relies on a common nuclear factor”. Nature 370:226,1994.). CBP in turn interacts with the basal transcription factor TFIIBcausing increased transcription. CREB also has been reported to interactwith dTAFII 110, a TATA binding protein-associated factor whose bindingmay regulate transcription (Ferreri, K., G. Gill, and M. Montminy: “ThecAMP-regulated transcription factor CREB interacts with a component ofthe TFIID complex”. Proc. Natl. Acad. Sci. USA 91:1210, 1994.). Inaddition to these interactions, CREB/ATF proteins can specifically bindmultiple other nuclear factors (Hoeffler, J. P., J. W. Lustbader, and C.-Y. Chen: “Identification of multiple nuclear factors that interact withcyclic adenosine 3′,5′-monophosphate response element-binding proteinand activating transcription factor-2 by protein-protein interactions”.Mol. Endocrinol. 5:256, 1991) but the biologic significance of most ofthese interactions is unknown. CREB is normally thought to bind DNAeither as a homodimer or as a heterodimer with several other proteins.Surprisingly, CREB monomers constitutively activate transcription(Krajewski, W., and K. A. W. Lee: “A monomeric derivative of thecellular transcription factor CREB functions as a constitutiveactivator”. Mol. Cell Biol. 14:7204, 1994.).

Aside from their critical role in regulating cellular transcription, ithas recently been shown that CREB/ATF proteins are subverted by someinfectious viruses and retroviruses, which require them for viralreplication. For example, the cytomegalovirus immediate early promoter,one of the strongest known mammalian promoters, contains eleven copiesof the CRE which are essential for promoter function (Chang, Y. -N., S.Crawford, J. Stall, D. R Rawlins, K. -T. Jeang, and G. S. Hayward: “Thepalindromic series I repeats in the simian cytomegalovirus majorimmediate-early promoter behave as both strong basal enhancers andcyclic AMP response elements”. J. Virol. 64:264, 1990). At least some ofthe transcriptional activating effects of the adenovirus E1A protein,which induces many promoters, are due to its binding to the DNA bindingdomain of the CREB/ATF protein, ATF-2, which mediates E1A inducibletranscription activation (Liu, F., and M. P Green: “Promoter targetingby adenovirus E1a through interaction with different cellularDNA-binding domains”. Nature 368:520, 1994). It has also been suggestedthat E1A binds to the CREB-binding protein, CBP (Arany, Z., W. R.Sellers, D. M. Livingston, and R. Eckner: “E1A-associated p300 andCREB-associated CBP belong to a conserved family of coactivators”. Cell77:799, 1994). Human T lymphotropic virus-I (HTLV-1), the retroviruswhich causes human T cell leukemia and tropical spastic paresis, alsorequires CREB/ATF proteins for replication. In this case, the retrovirusproduces a protein, Tax, which binds to CREB/ATF proteins and redirectsthem from their normal cellular binding sites to different DNA sequences(flanked by G- and C-rich sequences) present within the HTLVtranscriptional enhancer (Paca-Uccaralertkun, S., L. -J. Zhao, N. Adya,J. V. Cross, B. R Cullen, I. M. Boros, and C.-Z. Giam: “In vitroselection of DNA elements highly responsive to the human T-celllymphotropic virus type I transcriptional activator, Tax”. Mol. Cell.Biol. 14:456, 1994; Adya, N., L. -J. Zhao, W. Huang, I. Boros, and C.-Z. Giam: “Expansion of CREB's DNA recognition specificity by Taxresults from interaction with Ala-Ala-Arg at positions 282-284 near theconserved DNA-binding domain of CREB”. Proc. Natl. Acad. Sci. USA91:5642, 1994).

SUMMARY OF THE INVENTION

The instant invention is based on the finding that certainoligonucleotides containing unmethylated cytosine-guanine (CpG)dinucleotides activate lymphocytes as evidenced by in vitro and in vivodata. Based on this finding, the invention features, in one aspect,novel immunostimulatory oligonucleotide compositions.

In a preferred embodiment, an immunostimulatory oligonucleotide issynthetic, between 2 to 100 base pairs in size and contains a consensusmitogenic CpG motif represented by the formula:5′X₁X₂CGX₃X₄ 3′

-   -   wherein C and G are unmethylated, X₁, X₂, X₃ and X₄ are        nucleotides and a GCG trinucleotide sequence is not present at        or near the 5′ and 3′ termini.

For facilitating uptake into cells, CpG containing immunostimulatoryoligonucleotides are preferably in the range of 8 to 40 base pairs insize. Prolonged immunostimulation can be obtained using stabilizedoligonucleotides, particularly phosphorothioate stabilizedoligonucleotides. Enhanced immunostimulatory activity has been observedwhere X₁X₂ is the dinucleotide GpA and/or X₃X₄ is the dinucleotide ismost preferably TpC or also TpT. Further enhanced immunostimulatoryactivity has been observed where the consensus motif X₁X₂CGX₃X₄ ispreceded on the 5′ end by a T.

In a second aspect, the invention features useful methods, which arebased on the immunostimulatory activity of the oligonucleotides. Forexample, lymphocytes can either be obtained from a subject andstimulated ex vivo upon contact with an appropriate oligonucleotide; ora non-methylated CpG containing oligonucleotide can be administered to asubject to facilitate in vivo activation of a subject's lymphocytes.Activated lymphocytes, stimulated by the methods described herein (e.g.either ex vivo or in vivo), can boost a subject's immune response. Theimmunostimulatory oligonucleotides can therefore be used to treat,prevent or ameliorate an immune system deficiency (e.g., a tumor orcancer or a viral, fungal, bacterial or parasitic infection in asubject. In addition, immunostimulatory oligonucleotides can also beadministered as a vaccine adjuvant, to stimulate a subject's response toa vaccine. Further, the ability of immunostimulatory cells to induceleukemic cells to enter the cell cycle, suggests a utility for treatingleukemia by increasing the sensitivity of chronic leukemia cells andthen administering conventional ablative chemotherapy.

In a third aspect, the invention features neutral oligonucleotides (i.e.oligonucleotide that do not contain an unmethylated CpG or which containa methylated CpG dinucleotide). In a preferred embodiment, aneutralizing oligonucleotide is complementary to an immunostimulatorysequence, but contains a methylated instead of an unmethylated CpGdinucleotide sequence and therefore can compete for binding withunmethylated CpG containing oligonucleotides. In a preferred embodiment,the methylation occurs at one or more of the four carbons and twonitrogens comprising the cytosine six member ring or at one or more ofthe five carbons and four nitrogens comprising the guanine nine memberdouble ring. 5′ methyl cytosine is a preferred methylated CpG.

In a fourth aspect, the invention features useful methods using theneutral oligonucleotides. For example, in vivo administration of neutraloligonucleotides should prove useful for treating diseases such assystemic lupus erythematosus, sepsis and autoimmune diseases, which arecaused or exacerbated by the presence of unmethylated CpG dimers in asubject In addition, methylation CpG containing antisenseoligonucleotides or oligonucleotide probes would not initiate an immunereaction when administered to a subject in vivo and therefore would besafer than corresponding unmethylated oligonucleotides.

In a fifth aspect, the invention features immunoinhibitoryoligonucleotides, which are capable of interfering with the activity ofviral or cellular transcription factors. In a preferred embodiment,immunoinhibitory oligonucleotides are between 2to 100 base pairs in sizeand contain a consensus immunoinhibitory CpG motif represented by theformula:⁵′GCGXnGCG³′

-   -   wherein X=a nucleotide and n=in the range of 0-50. In a        preferred embodiment, X is a pyrimidine.

For facilitating uptake into cells, immunoinhibitory oligonucleotidesare preferably in the range of 8 to 40 base pairs in size. Prolongedimmunostimulation can be obtained using stabilized oligonucleotides,particularly phosphorothioate stabilized oligonucleotides.

In a sixth and final aspect, the invention features various uses forimmunoinhibitory oligonucleotides. Immunoinhibitory oligonucleotideshave antiviral activity, independent of any antisense effect due tocomplementarity between the oligonucleotide and the viral sequence beingtargeted.

Other features and advantages of the invention will become more apparentfrom the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below:

An “oligonucleotide” or “oligo” shall mean multiple nucleotides (i.e.molecules comprising a sugar (e.g. ribose or deoxyribose) linked to aphosphate group and to an exchangeable organic base, which is either asubstituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) ora substituted purine (e.g. adenine (A) or guanine (G)). The term“oligonucleotide” as used herein refers to both oligoribonucleotides(ORNs) and oligodeoxyribonucleotides (ODNs). The term. “oligonucleotide”shall also include oligonucleosides (i.e. an oligonucleotide minus thephosphate) and any other organic base containing polymer.Oligonucleotides can be obtained from existing nucleic acid sources(e.g. genomic or cDNA), but are preferably synthetic (e.g. produced byoligonucleotide synthesis).

A “stabilized oligonucledtide” shall mean an oligonucleotide that isrelatively resistant to in vivo degradation (e.g. via an exo- orendo-nuclease). Preferred stabilized oligonucleotides of the instantinvention have a modified phosphate backbone. Especially preferredoligonucleotides have a phosphorothioate modified phosphate backbone(i.e. at least one of the phosphate oxygens is replaced by sulfur).Other stabilized oligonucleotides include: nonionic DNA analogs, such asakyl- and aryl- phosphonates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Oligonucleotides which contain a diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

An “immunostimulatory oligonucleotide”, “immunostimulatory CpGcontaining oligonucleotide”, or “CpG ODN” refer to an oligonucleotide,which contains a cytosine, guanine dinucleotide sequence and stimulates(e.g. has a mitogenic effect) on vertebrate lymphocyte. Preferredimmunostimulatory oligonucleotides are between 2 to 100 base pairs insize and contain a consensus mitogenic CpG motif represented by theformula:^(5′)X₁X₂CGX₃X₄ ³′

-   -   wherein C and G are unmethylated, X₁, X₂, X₃ and X₄ are        nucleotides and a GCG trinucleotide sequence is not present at        or near the 5′ and 3′ termini.

Preferably the immunostimulatory oligonucleotides range between 8 to 40base pairs in size. In addition, the immunostimulatory oligonucleotidesare preferably stabilized oligonucleotides, particularly preferred arephosphorothioate stabilized oligonucleotides. In one preferredembodiment, X₁X₂ is the dinucleotide GpA. In another preferredembodiment, X₃X₄ is preferably the dinucleotide TpC or also TpT. In aparticularly preferred embodiment, the consensus motif X₁X₂CGX₃X₄ ispreceded on the 5′ end by a T. Particularly preferred consensussequences are TGACGTT or TGACGTC.

A “neutral oligonucleotide” refers to an oligonucleotide that does notcontain an unmethylated CpG or an oligonucleotide which contains amethylated CpG dinucleotide. In a preferred embodiment, a neutralizingoligonucleotide is complementary to an immunostimulatory sequence, butcontains a methylated instead of an unmethylated CpG dinucleotidesequence and therefore can compete for binding with unmethylated CpGcontaining oligonucleotides. In a preferred embodiment, the methylationoccurs at one or more of the four carbons and two nitrogens comprisingthe cytosine six member ring or at one or more of the five carbons andfour nitrogens comprising the guanine nine member double ring. 5′ methylcytosine is a preferred methylated CpG.

An “immunoinhibitory oligonucleotide” or “immunoinhibitory CpGcontaining oligonucleotide” is an oligonucleotide that. Preferableimmunoinhibitory oligonucleotides are between 2 to 100 base pairs insize and can be represented by the formula:^(5′)GCGXnGCG^(3′)

-   -   wherein X=a nucleotide and n=in the range of 0-50. In a        preferred embodiment, X is a pyrimidine.

For facilitating uptake into cells, immunoinhibitory oligonucleotidesare preferably in the range of 8 to 40 base pairs in size. Prolongedimmunostimulation can be obtained using stabilized oligonucleotides,particularly phosphorothioate stabilized

“Palindromic sequence” shall mean an inverted repeat (i.e. a sequencesuch as ABCDEE′D′C′B′A′ in which A and A′ are bases capable of formingthe usual Watson-Crick base pairs. In vivo, such sequences may formdouble stranded structures.

An “oligonucleotide delivery complex” shall mean an oligonucleotideassociated with (e.g. ionically or covalently bound to; or encapsulatedwithin) a targeting means (e.g. a molecule that results in higheraffinity binding to target cell (e.g. B-cell and natural killer (NK)cell) surfaces and/or increased cellular uptake by target cells).Examples of oligonucleotide delivery complexes include oligonucleotidesassociated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationiclipid, virosome or liposome), or a target cell specific binding agent(e.g. a ligand recognized by target cell specific receptor). Preferredcomplexes must be sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex should be cleavable under appropriate conditions within the cellso that the oligonucleotide is released in a functional form.

An “immune system deficiency” shall mean a disease or disorder in whichthe subject's immune system is not functioning in normal capacity or inwhich it would be useful to boost a subject's immune response forexample to eliminate a tumor or cancer (e.g. tumors of the brain, lung(e.g. small cell and non-small cell), ovary, breast prostate, colon, aswell as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes),fungal (e.g. Candida sp.), bacterial or parasitic (e.g. Leishmania,Toxoplasma) infection in a subject

A “disease associated with immune system activation” shall mean adisease or condition caused or exacerbated by activation of thesubject's immune system. Examples include systemic lupus erythematosus,sepsis and autoimmune diseases such as rheumatoid arthritis and multiplesclerosis.

A “subject” shall mean a human or vertebrate animal including a dog,cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, mouse, etc.

Certain Unmethylated CpG Containing Oligos have B Cell StimulatoryActivity as Shown In Vitro and In Vivo

In the course of investigating the lymphocyte stimulatory effects of twoantisense oligonucleotides specific for endogenous retroviral sequences,using protocols described in the attached Examples 1 and 2, it wassurprisingly found that two out of twenty-four “controls” (includingvarious scrambled, sense, and mismatch controls for a panel of“antisense” ODN) also mediated B cell activation and IgM secretion,while the other “controls” had no effect

Two observations suggested that the mechanism of this B cell activationby the “control” ODN may not involve antisense effects 1) comparison ofvertebrate DNA sequences listed in GenBank showed no greater homologythan that seen with non-stimulatory ODN and 2) the two controls showedno hybridization to Northern blots with 10 μg of spleen poly A+ RNA.Resynthesis of these ODN on a different synthesizer or extensivepurification by polyacrylamide gel electrophoresis or high pressureliquid chromatography gave identical stimulation, eliminating thepossibility of an impurity. Similar stimulation was seen using B cellsfrom C3H/HeJ mice, eliminating the possibility that lipopolysaccharide(LPS) contamination could account for the results.

The fact that two “control” ODN caused B cell activation similar to thatof the two “antisense” ODN raised the possibility that all four ODN werestimulating B cells through some non-antisense mechanism involving asequence motif that was absent in all of the other nonstimulatorycontrol ODN. In comparing these sequences, it was/discovered that all ofthe four stimulatory ODN contained ODN dinucleotides that were in adifferent sequence context from the nonstimulatory control

To determine whether the CpG motif present in the stimulatory ODN wasresponsible for the observed stimulation, over 300 ODN ranging in lengthfrom 5 to 42 bases that contained methylated, unmethylated, or no CpGdinucleotides in various sequence contexts were synthesized. These ODNs,including the two original “controls” (ODN 1 and 2) and two originallysynthesized as “antisense” (ODN 3D and 3M; Krieg; A. M. J. Immunol.143:2448 (1989)), were then examined for in vitro effects on spleencells (representative sequences are listed in Table 1). Several ODN thatcontained CpG dinucleotides induced B cell activation and IgM secretion;the magnitude of this stimulation typically could be increased by addingmore CpG dinucleotides (Table 1; compare ODN 2 to 2a or 3D to 3Da and 3Db). Stimulation did not appear to result from an antisense mechanism orimpurity. ODN caused no detectable activation of γδ or other T cellpopulations.

Mitogenic ODN sequences uniformly became nonstimulatory if the CpGdinucleotide was mutated (Table 1; compare ODN 1 to 1a; 3D to. 3Dc; 3Mto 3Ma; and 4 to 4a) or if the cytosine of the CpG dinucleotide wasreplaced by 5-methylcytosine (Table 1; ODN 1b, 2b, 2c, 3Dd, and 3 Mb).In contrast, methylation of other cytosines did not reduce ODN activity(ODN 1c, 2d, 3De and 3Mc). These data confirmed that a CpG motif is theessential element present in ODN that activate B cells.

In the course of these studies, it became clear that the bases flankingthe CpG dinucleotide played an important role in determining the B cellactivation induced by an ODN. The optimal stimulatory motif wasdetermined to consist of a CpG flanked by two 5′ purines (preferably aGpA dinucleotide) and two 3′ pyrimidines (preferably a TpT or TpCdinucleotide). Mutations of ODN to bring the CpG motif closer to thisideal improved stimulation (e.g. compare ODN 2 to 2e; 3M to 3Md) whilemutations that disturbed the motif reduced stimulation (e.g. compare ODN3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutations outside theCpG motif did not reduce stimulation (e.g. compare ODN 1 to 1d; 3D to3Dg; 3M to 3Me).

Of those tested, ODNs shorter than 8 bases were non-stimulatory (e.g.ODN 4e). Among the forty-eight 8 base ODN tested, the most stimulatorysequence identified was TCAACGTT (ODN 4) which contains the selfcomplementary “palindrome” AACGTT. In further optimizing this motif, itwas found that ODN containing Gs at both ends showed increasedstimulation, particularly if the the ODN were rendered nucleaseresistant by phosphorothioate modification of the terminalinternucleotide linkages. ODN 1585 (5′ GGGGTCAACGTTCAGGGGGG 3′ (SEQ IDNO:1)), in which the first two and last five internucleotide linkagesare phosphorothioate modified caused an average 25.4 fold increase inmouse spleen cell proliferation compared to an average 3.2 fold increasein proliferation induced by ODN 1638, which has the same sequence as ODN1585 except that the 10 Gs at the two ends are replaced by 10 As. Theeffect of the G-rich ends is cis; addition of an ODN with poly G endsbut no CpG motif to cells along with 1638 gave no increasedproliferation.

Other octamer ODN containing a 6 base palindrome with a TpC dinucleotideat the 5′ end were also active if they were close to the optimal motif(e.g. ODN 4b,4c). Other dinucleotides at the 5′ end gave reducedstimulation (eg ODN 4f; all sixteen possible dinucleotides were tested).The presence of a 3′ dinucleotide was insufficient to compensate for thelack of a 5′ dinucleotide (eg. ODN. 4g). Disruption of the palindromeeliminated stimulation in octamer ODN (eg., ODN 4h), but palindromeswere not required in longer ODN. TABLE 1 Oligonucleotide Stimulation ofB Cells Stimulation Index' ODN Sequence (5′ to 3′)† ³H Uridine IgMProduction 1 (SEQ ID NO: 2) GCTAGACGTTAGCGT 6.1 ± 0.8 17.9 ± 3.6  1a(SEQ. ID NO: 3) ......T........ 1.2 ± 0.2 1.7 ± 0.5 1b (SEQ ID NO: 4)......Z........ 1.2 ± 0.1 1.8 ± 0.0 1c (SEQ ID NO: 5) ............Z..10.3 ± 4.4  9.5 ± 1.8 1d (SEQ ID NO: 6) ..AT......GAGC. 13.0 ± 2.3  18.3± 7.5  2 (SEQ ID NO: 7) ATGGAAGGTCCAGCGTTCTC 2.9 ± 0.2 13.6 ± 2.0  2a(SEQ ID NO: 8) ..C..CTC..G......... 7.7 ± 0.8 24.2 ± 3.2  2b (SEQ ID NO:9) ..Z..CTC.ZG..Z...... 1.6 ± 0.5 2.8 ± 2.2 2c (SEQ ID NO: 10)..Z..CTC..G......... 3.1 ± 0.6 7.3 ± 1.4 2d (SEQ ID NO: 11)..C..CTC..G......Z.. 7.4 ± 1.4 27.7 ± 5.4  2e (SEQ ID NO: 12)............A....... 5.6 ± 2.0 ND 3D (SEQ ID NO: 13)GAGAACGCTGGACCTTCCAT 4.9 ± 0.5 19.9 ± 3.6  3Da (SEQ ID NO: 14).........C.......... 6.6 ± 1.5 33.9 ± 6.8  3Db (SEQ ID NO: 15).........C.......G.. 10.1 ± 2.8  25.4 ± 0.8  3Dc (SEQ ID NO: 16)...C.A.............. 1.0 ± 0.1 1.2 ± 0.5 3Dd (SEQ ID NO: 17).....Z.............. 1.2 ± 0.2 1.0 ± 0.4 3De (SEQ ID NO: 18).............Z...... 4.4 ± 1.2 18.8 ± 4.4  3Df (SEQ ID NO: 19).......A............ 1.6 ± 0.1 7.7 ± 0.4 3Dg (SEQ ID NO: 20).........CC.G.ACTG.. 6.1 ± 1.5 18.6 ± 1.5  3M (SEQ ID NO: 21)TCCATGTCGGTCCTGATGCT 4.1 ± 0.2 23.2 ± 4.9  3Ma (SEQ ID NO: 22)......CT............ 0.9 ± 0.1 1.8 ± 0.5 3Mb (SEQ ID NO: 23).......Z............ 1.3 ± 0.3 1.5 ± 0.6 3Mc (SEQ ID NO: 24)...........Z........ 5.4 ± 1.5 8.5 ± 2.6 3Md (SEQ ID NO: 25)......A..T.......... 17.2 ± 9.4  ND 3Me (SEQ ID NO: 26)...............C..A. 3.6 ± 0.2 14.2 ± 5.2  4 TCAACGTT 6.1 ± 1.4 19.2± 5.2  4a ....GC.. 1.1 ± 0.2 1.5 ± 1.1 4b ...GCGC. 4.5 ± 0.2 9.6 ± 3.44c ...TCGA. 2.7 ± 1.0 ND 4d ..TT..AA 1.3 ± 0.2 ND 4e -....... 1.3 ± 0.21.1 ± 0.5 4f C....... 3.9 ± 1.4 ND 4g --......CT 1.4 ± 0.3 ND 4h.......C 1.2 ± 0.2 ND LPS 7.8 ± 2.5 4.8 ± 1.0'Stimulation indexes are the means and std. dev. derived from at least 3separate experiments, and are compared to wells cultured with no addedODN.ND = not done.CpG dinucleotides are underlined.Dots indicate identity; dashes indicate deletions.Z indicates 5 methyl cytosine.)

The kinetics of lymphocyte activation were investigated using mousespleen cells. When the cells were pulsed at the same time as ODNaddition and harvested just four hours later, there was already atwo-fold increase in ³H uridine incorporation. Stimulation peaked at12-48 hours and then decreased. After 24 hours, no intact ODN weredetected, perhaps accounting for the subsequent fall in stimulation whenpurified B cells with or without anti-IgM (at a submitogenic dose) werecultured with CpG ODN, proliferation was found to synergisticallyincrease about 10-fold by the two mitogens in combination after 48hours. The magnitude of stimulation was concentration dependent andconsistently exceeded that of LPS under optimal conditions for both.Oligonucleotides containing a nuclease resistant phosphorothioatebackbone were approximately two hundred times more potent thanunmodified oligonucleotides.

Cell cycle analysis was used to determine the proportion of B cellsactivated by CpG-ODN. CpG-ODN induced cycling in more than 95% of Bcells (Table 2). Splenic B lymphocytes sorted by flow cytometry intoCD23− (marginal zone) and CD23+ (follicular) subpopulations were equallyresponsive to ODN-induced stimulation, as were both resting andactivated populations of B cells isolated by fractionation over Percollgradients. These studies demonstrated that CpG-ODN induce essentiallyall B cells to enter the cell cycle. TABLE 2 Cell Cycle Analysis withCpG ODN Percent of cells in Treatment G0 G1 SA + G2 + M Media 97.6 2.40.02 ODN 1a 95.2 4.8 0.04 ODN 1d 2.7 74.4 22.9 ODN 3Db 3.5 76.4 20.1 LPS(30 μg/ml) 17.3 70.5 12.2

The mitogenic effects of CpG ODN on human cells, were tested onperipheral blood mononuclear cells (PBMCs) obtained from two patientswith chronic lymphocytic leukemia (CLL), as described in Example 1.Control ODN containing no CpG dinucleotide sequence showed no effect onthe basal proliferation of 442 cpm and 874 cpm (proliferation measuredby ³H thymidine incorporation) of the human cells. However, aphosphorothioate modified CpG ODN 3Md (SEQ ID NO: 25) induced increasedproliferation of 7210 and 86795 cpm respectively in the two patients ata concentration of just 1 μM. Since these cells had been frozen, theymay have been less responsive to the oligos than fresh cells in vivo. Inaddition, cells from CLL patients typically are non-proliferating, whichis why traditional chemotherapy is not effective.

Certain B cell lines such as WEHI-231 are induced to undergo growtharrest and/or apoptosis in response to crosslinking of their antigenreceptor by anti-IgM (Jakway, J. P. et al., “Growth regulation of the Blymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharideand other bacterial products” J. Immunol. 137: 2225 (1986); Tsubata, T.,J. Wu and T. Honjo: B-cell apoptosis induced by antigen receptorcrosslinking is blocked by a T-cell signal through CD40.” Nature 364:645 (1993)). WEHI-231 cells are rescued from this growth arrest bycertain stimuli such as LPS and by the CD40 ligand. ODN containing theCpG motif were also found to protect WEHI-231 from anti-IgM inducedgrowth arrest, indicating that accessory cell populations are notrequired for the effect.

To better understand the immune effects of unmethylated CpG ODN, thelevels of cytokines and prostaglandins in vitro and in vivo weremeasured. Unlike LPS, CpG ODN were not found to induce purifiedmacrophages to produce prostaglandin PGE2. In fact, no apparent directeffect of CpG ODN was detected on either macrophages or T cells. In vivoor in whole spleen cells, no significant increase in the followinginterleukins: IL-2, IL-3, IL4, or IL-10 was detected within the firstsix hours. However, the level of IL-6 increased strikingly within 2hours in the serum of mice injected with CpG ODN. Increased expressionof IL-12 and interferon gamma (IFN-γ) by spleen cells was also detectedwithin the first two hours.

To determine whether CpG ODN can cause in vivo immune stimulation, DBA/2mice were injected once intraperitoneally with PBS or phosphorothioateCpG or non-CpG ODN at a dose of 33 mg/kg (approximately 500 μg/mouse).Pharmacokinetic studies in mice indicate that this dose ofphosphorothioate gives levels of approximately 10 μg/g in spleen tissue(within the effective concentration range determined from the in vitrostudies described herein) for longer than twenty-four-hours (Agrawal, S.et al. (1991) Proc. Natl. Acad. Sci. USA 91:7595). Spleen cells frommice were examined twenty-four hours after ODN injection for expressionof B cells surface activation markers Ly-6A/E, Bla-1, and class II MHCusing three color flow cytometry and for their spontaneous proliferationusing ³H thymidine: Expression of all three activation markers wassignificantly increased in B cells from mice injected with CpG ODN, butnot from mice injected with PBS or non-CpG ODN. Spontaneous ³H thymidineincorporation was increased by 2-6 fold in spleen cells from miceinjected with the stimulatory ODN compared to PBS or non-CpGODN-injected mice. After 4 days, serum IgM levels in mice injected withCpG ODN in vivo were increased by approximately 3-fold compared tocontrols. Consistent with the inability of these agents to activate Tcells, there was minimal change in T cell expression of the IL-2R orCD44.

Degradation of phophodiester ODN in serum is predominantly mediated by3′ exonucleases, while intracellular ODN degradation is more complex,involving 5′ and 3′ exonucleases and endonucleases. Using a panel of ODNbearing the 3D sequence with varying numbers of phosphorothioatemodified linkages at the 5′ and 3′ ends, it was empirically determinedthat two 5′ and five 3′ modified linkages are required to provideoptimal stimulation with this CpG ODN.

Unmethylated CpG Containing Oligos have NK Cell Stimulatory Activity

As described in further detail in Example 4, experiments were conductedto determine whether CpG containing oligonucleotides stimulated theactivity of natural killer (NK) cells in addition to B cells. As shownin Table 3, a marked induction of NK activity among spleen cellscultured with CpG ODN 1 and 3Dd was observed. In contrast, there %% asrelatively no induction in effectors that had been treated with non-CpGcontrol ODN. TABLE 3 Induction Of NK Activity By CpGOligodeoxynucleotides (ODN) % YAC-1 Specific Lysis* % 2C11 SpecificLysis Effector:Target Effector:Target ODN 50:1 100:1 50:1 100:1 None−1.1 −1.4 15.3 16.6 1 16.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0non-CpG ODN −1.6 −1.7 14.8 15.4

Neutralizing Activity of Methylated CpG Containing Oligos

B cell mitogenicity of ODN in which cytosines in CpG motifs or elsewherewere replaced by 5-methylcytosine were tested as described in Example 1.As shown in Table 1 above, ODN containing methylated CpG motifs werenon-mitogenic (Table 1; ODN 1c, 2f, 3De, and 3Mc). However, methylationof cytosines other than in a CpG dinucleotide retained their stimulatoryproperties (Table 1, ODN 1d, 2d, 3Df, and 3Md).

Immunoinhibitory Activity of Oligos Containing a GCG TrinucleotideSequence at or Near Both Termini

In some cases, ODN containing CpG dinucleotides that are not in thestimulatory motif described above were found to block the stimulatoryeffect of other mitogenic CpG ODN. Specifically the addition of anatypical CpG motif consisting of a GCG near or at the 5′ and/or 3′ endof CpG ODN actually inhibited stimulation of proliferation by other CpGmotifs. Methylation or substitution of the cytosine in a GCG motifreverses this effect. By itself, a GCG motif in an ODN has a modestmitogenic effect, though far lower than that seen with the preferred CpGmotif.

Proposed Mechanisms of Action of Immunostimulatory, Neutralizing andImmunoinhibitory Oligonucleotides

Unlike antigens that trigger B cells through their surface Ig receptor,CpG-ODN did not induce any detectable Ca²⁺ flux, changes in proteintyrosine phosphorylation, or IP 3 generation. Flow cytometry withFITC-conjugated ODN with or without a CpG motif was performed asdescribed in Zhao, Q et al., (Antisense Research and Development 3:53-66(1993)), and showed equivalent membrane binding, cellular uptake,efflux, and intracellular localization. This suggests that there may notbe cell membrane proteins specific for CpG ODN. Rather than actingthrough the cell membrane, that data suggests that unmethylated CpGcontaining oligonucleotides require cell uptake for activity: ODNcovalently linked to a solid Teflon support were nonstimulatory, as werebiotinylated ODN immobilized on either avidin beads or avidin coatedpetri dishes. CpG ODN conjugated to either FITC or biotin retained fullmitogenic properties, indicating no steric hindrance.

The optimal CpG motif (TGACGTT/C is identical to the CRE (cyclic AMPresponse element). Like the mitogenic effects of CpG ODN, binding ofCREB to the CRE is abolished if the central CpG is methylated.Electrophoretic mobility shift assays were used to determine whether CpGODN, which are single stranded, could compete with the binding of B cellCREB/ATF proteins to their normal binding site, the doublestranded CRE.Competition assays demonstrated that single stranded ODN containing CpGmotifs could completely compete the binding of CREB to its binding site,while ODN without CpG motifs could not. These data support theconclusion that CpG ODN exert their mitogenic effects throughinteracting with one or more-B cell CREB/ATF proteins in some way.Conversely, the presence of GCG sequences or other atypical CPG motifsnear the 5′ and/or 3′ ends of ODN likely interact with CREB/ATF proteinsin away that does not cause activation, and may even prevent it.

The stimulatory CpG motif is common in microbial genomic DNA, but quiterare in vertebrate DNA. In addition, bacterial DNA has been reported toinduce B cell proliferation and immunoglobulin (Ig) production, whilemammalian DNA does not (Messina, J. P. et al., J. Immunol. 147:1759(1991)). Experiments further described in Example 3, in whichmethylation of bacterial DNA with CpG methylase was found to abolishmitogenicity, demonstrates that the difference in CpG status is thecause of B cell stimulation by bacterial DNA. This data supports thefollowing conclusion: that unmethylated CpG dinucleotides present withinbacterial DNA are responsible for the stimulatory effects of bacterialDNA.

Teleologically, it appears likely that lymphocyte activation by the CpGmotif represents an immune defense mechanism that can therebydistinguish bacterial from host DNA. Host DNA would induce little or nolymphocyte activation due to it CpG suppression and methylation.Bacterial DNA would cause selective lymphocyte activation in infectedtissues. Since the CpG pathway synergizes with B cell activation throughthe antigen receptor, B cells bearing antigen receptor specific forbacterial antigens would receive one activation signal through cellmembrane Ig and a second signal from bacterial DNA, and would thereforetend to be preferentially activated. The interrelationship of thispathway with other pathways of B cell activation provide a physiologicmechanism employing a polyclonal antigen to induce antigen-specificresponses.

Method for Making Immunostimulatory Oligos

For use in the instant invention, oligonucleotides can be synthesized denovo using any of a number of procedures well known in the art. Forexample, the β-cyanoethyl phosphoramidite method (S. L. Beaucage and M.H. Caruthers, (1981) Tet. Let. 22:1859); nucleoside H-phosphonate method(Garegg-et al., (1986) Tet. Let. 27: 4051-4054; Froehler et al., (1986)Nucl. Acid Res. 14: 5399-5407; Garegg et al., (1986) Tet. Let. 27:4055-4058, Gaffney et al., (1988) Tet. Let. 29:2619-2622). Thesechemistries can be performed by a variety of automated oligonucleotidesynthesizers available in the market. Alternatively, oligonucleotidescan be prepared from existing nucleic acid sequences (e.g. genomic orcDNA) using known techniques, such as those employing restrictionenzymes, exonucleases or endonucleases.

For use in vivo, oligonucleotides are preferably relatively resistant todegradation (e.g. via endo- and exo- nucleases). Oligonucleotidestabilization can be accomplished via phosphate backbone modifications.A preferred stabilized oligonucleotide has a phosphorothioate modifiedbackbone. The pharmacokinetics of phosphorothioate ODN show that theyhave a systemic half-life of forty-eight hours in rodents and suggestthat they may be useful for in vivo applications (Agrawal, S. et al.(1991) Proc. Natl. Acad. Sci. USA 88:7595). Phosphorothioates may besynthesized using automated techniques employing either phosphoramidateor H phosphonate chemistries. Aryl- and alkyl- phosphonates can be madee.g. (as described in U.S. Pat. No. 4,469,863); andalkylphosphotriesters (in which the charged oxygen moiety is alkylatedas described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574)can be prepared by automated solid phase synthesis using commerciallyavailable reagents. Methods for making other DNA backbone modificationsand substitutions have been described Uhlmann, E. and Peyman, A (1990)Chem. Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165).

For administration in vivo, oligonucleotides may be associated with amolecule that results in higher affinity binding to target cell (e.g.B-cell and natural killer (NK) cell) surfaces and/or increased cellularuptake by target cells to form an “oligonucleotide delivery complex”.Oligonucleotides can be ionically, or covalently associated withappropriate molecules using techniques which are well known in the art.A variety of coupling or crosslinking agents can be used e.g. protein A,carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP).Oligonucleotides can alternatively be encapsulated in liposomes orvirosomes using well-known techniques.

The present invention is further illustrated by the following Exampleswhich in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Therapeutic Uses of Immunostimulatory Oligos

Based on their immunostimulatory properties, oligonucleotides containingat least one unmethylated CpG dinucleotide can be administered to asubject in vivo to treat an “immune system deficiency”. Alternatively,oligonucleotides containing at least one unmethylated CpG dinucleotidecan be contacted with lymphocytes (e.g. B cells or NK cells) obtainedfrom a subject having an immune system deficiency ex vivo and activatedlymphocytes can then be reimplanted in the subject

Immunostimulatory oligonucleotides can also be administered to a subjectin conjunction with a vaccine, as an adjuvant, to boost a subject'simmune system to effect better response from the vaccine. Preferably theunmethylated CpG dinucleotide is administered slightly before or at thesame time as the vaccine.

Preceding chemotherapy with an immunostimulatory oligonucleotide shouldprove useful for increasing the responsiveness of the malignant cells tosubsequent chemotherapy. CpG ODN also increased natural killer cellactivity in both human and murine cells. Induction of NK activity maylikewise be beneficial in cancer immunotherapy.

Therapeutic Uses for Neutral Oligonucleotides

Oligonucleotides that are complementary to certain target sequences canbe synthesized and administered to a subject in vivo. For example,antisense oligonucleotides hybridize to complementary mRNA, therebypreventing expression of a specific target gene. The sequence-specificeffects of antisense oligonucleotides have made them useful researchtools for the investigation of protein function. Phase I/II human trialsof systemic antisense therapy are now underway for acute myelogenousleukemia and HIV.

In addition, oligonucleotide probes (i.e. oligonucleotides with adetectable label) can be administered to a subject to detect thepresence of a complementary sequence based on detection of bound label.In vivo administration and detection of oligonucleotide probes may beuseful for diagnosing certain diseases that are caused or exacerbated bycertain DNA sequences (e.g. systemic lupus erythematosus, sepsis andautoimmune diseases).

Antisense oligonucleotides or oligonucleotide probes in which any or allCpG dinucleotide is methylated, would not produce an immune reactionwhen administered to a subject in vivo and therefore would be safer thanthe corresponding non-methylated CpG containing oligonucleotide.

For use in therapy, an effective amount of an appropriateoligonucleotide alone or formulated as an oligonucleotide deliverycomplex can be administered to a subject by any mode allowing theoligonucleotide to be taken up by the appropriate target cells (e.g.B-cells and NK cells). Preferred routes of administration include oraland transdermal (e.g. via a patch). Examples of other routes ofadministration include injection (subcutaneous, intravenous, parenteral,intraperitoneal, intrathecal, etc.). The injection can be in a bolus ora continuous infusion.

An oligonucleotide alone or as an oligonucleotide delivery complex canbe administered in conjunction with a pharmaceutically acceptablecarrier. As used herein, the phrase “pharmaceutically acceptablecarrier” is intended to include substances that can be coadministeredwith an oligonucleotide or an oligonucleotide delivery complex andallows the oligonucleotide to perform its intended function. Examples ofsuch carriers include solutions, solvents, dispersion media, delayagents, emulsions and the like. The use of such media forpharmaceutically active substances are well known in the art. Any otherconventional carrier suitable for use with the oligonucleotides fallswithin the scope of the instant invention.

The language “effective amount” of an oligonucleotide refers to thatamount necessary or sufficient to realize a desired biologic effect. Forexample, an effective amount of an oligonucleotide containing at leastone methylated CpG for treating an immune system deficiency could bethat amount necessary to eliminate a tumor, cancer, or bacterial, viralor fungal infection. An effective amount for use as a vaccine adjuvantcould be that amount useful for boosting a subject's immune response toa vaccine. An “effective amount” of an oligonucleotide lacking anon-methylated CpG for use in treating a disease associated with immunesystem activation, could be that amount necessary to outcompetenon-methylated CpG containing nucleotide sequences. The effective amountfor any particular application can vary depending on such factors as thedisease or condition being treated, the particular oligonucleotide beingadministered, the size of the subject, or the severity of the disease orcondition. One of ordinary skill in the art can empirically determinethe effective amount of a particular oligonucleotide withoutnecessitating undue experimentation.

The studies reported above indicate that unmethylated CpG containingoligonucleotides are directly mitogenic for lymphocytes (e.g. B cellsand NK cells). Together with the presence of these sequences inbacterial DNA, these results suggest that the underrepresentation of CpGdinucleotides in animal genomes, and the extensive methylation ofcytosines present in such dinucleotides, may be explained by theexistence of an immune defense mechanism that can distinguish bacterialfrom host DNA. Host DNA would commonly be present in many anatomicregions and areas of inflammation due to apoptosis (cell death), butgenerally induces little or no lymphocyte activation. However, thepresence of bacterial DNA containing unmethylated CpG motifs can causelymphocyte activation precisely in infected anatomic regions, where itisbeneficial. This novel activation pathway provides a rapid alternativeto T cell dependent antigen specific B cell activation. However, it islikely that B cell activation would not be totally nonspecific. B cellsbearing antigen receptors specific for bacterial products could receiveone activation signal through cell membrane Ig, and a second frombacterial DNA, thereby more vigorously triggering antigen specificimmune responses.

As with other immune defense mechanisms, the response to bacterial DNAcould have undesirable consequences in some settings. For example,autoimmune responses to self antigens would also tend to bepreferentially triggered by bacterial infections, since autoantigenscould also provide a second activation signal to autoreactive B cellstriggered by bacterial DNA. Indeed the induction of autoimmunity bybacterial infections is a common clinical observance. For example, theautoimmune disease systemic lupus erythematosus, which is: i)characterized by the production of anti-DNA antibodies; ii) induced bydrugs which inhibit DNA methyltransferase (Cornacchia, E. J. et al., J.Clin Invest. 92:38 (1993)); and iii) associated with reduced DNAmethylation (Richardson, B., L. et al., Arth. Rheum 35:647 (1992)), islikely triggered at least in part by activation of DNA-specific B cellsthrough stimulatory signals provided by CpG motifs, as well as bybinding of bacterial DNA to antigen receptors.

Further, sepsis, which is characterized by high morbidity and mortalitydue to massive and nonspecific activation of the immune system may beinitiated by bacterial DNA and other products released from dyingbacteria that reach concentrations sufficient to directly activate manylymphocytes.

Lupus, sepsis and other “diseases associated with immune systemactivation” may be treated, prevented or ameliorated by administering toa subject oligonucleotides lacking an unmethylated CpG dinucleotide(e.g. oligonucleotides that do not include a CpG motif oroligonucleotides in which the CpG motif is methylated) to block thebinding of unmethylated CpG containing nucleic acid sequences.Oligonucleotides lacking an unmethylated CpG motif can be administeredalone or in conjunction with compositions that block an immune cell'sreponse to other mitogenic bacterial products (e.g. LPS).

The following serves to illustrate mechanistically how oligonucleotidescontaining an unmethylated CpG dinucleotide can treat, prevent orameliorate the disease lupus. Lupus is commonly thought to be triggeredby bacterial or viral infections. Such infections have been reported tostimulate the production of nonpathogenic antibodies to single strandedDNA. These antibodies likely recognize primarily bacterial sequencesincluding unmethylated CpGs. As disease develops in lupus, the anti-DNAantibodies shift to pathogenic antibodies that are specific fordouble-stranded DNA. These antibodies would have increased binding formethylated CpG sequences and their production would result from abreakdown of tolerance in lupus. Alternatively, lupus may result when apatient's DNA becomes hypomethylated, thus allowing anti-DNA antibodiesspecific for unmethylated CpGs to bind to self DNA and trigger morewidespread autoimmunity through the process referred to as “epitopespreading”.

In either case, it may be possible to restore tolerance in lupuspatients by coupling antigenic oligonucleotides to a protein carriersuch as gamma globulin (IgG). Calf-thymus DNA complexed to gammaglobulin has been reported to reduce anti-DNA antibody formation.

Therapeutic Uses of Oligos Containing GCG Trinucleotide Sequences at orNear Both Termini

Based on their interaction with CREB/ATF, oligonucleotides containingGCG trinucleotide sequences at or near both termini have antiviralactivity, independent of any antisense effect due to complementaritybetween the oligonucleotide and the viral sequence being targeted. Basedon this activity, an effective amount of inhibitory oligonucleotides canbe administered to a subject to treat or prevent a viral infection.

EXAMPLES Example 1 Effects of ODNs on B Cell Total RNA Synthesis andCell Cycle

B cells were purified from spleens obtained from 6-12 wk old specificpathogen free DBA/2 or BXSB mice (bred in the University of Iowa animalcare facility; no substantial strain differences were noted) that weredepleted of T cells with anti-Thy-1.2 and complement and centrifugationover lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) (“Bcells”). B cells contained fewer than 1% CD4⁺ or CD8⁺ cells. 8×10⁴ Bcells were dispensed in triplicate into 96 well microtiter plates in 100μl RPMI containing 10% FBS (heat inactivated to 65° C. for 30 min.), 50μM 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2mM L-glutamate. 20 μM ODN were added at the start of culture for 20 h at37° C., cells pulsed with 1 μCi of ³H uridine, and harvested and counted4 hr later. Ig secreting B cells were enumerated using the ELISA spotassay after culture of whole spleen cells with ODN at 20 μM for 48 hr.Data, reported in Table 1, represent the stimulation index compared tocells cultured without ODN. Cells cultured without ODN gave 687 cpm,while cells cultured with 20 μg/ml LPS (determined by titration to bethe optimal concentration) gave 99,699 cpm in this experiment. ³Hthymidine incorporation assays showed similar results, but with somenonspecific inhibition by thymidine released from degraded ODN (Matson.S and A. M. Krieg (1992) Nonspecific suppression of ³H-thymidineincorporation by control oligonucleotides. Antisense Research andDevelopment 2:325).

For cell cycle analysis, 2×10⁶ B cells were cultured for 48 hr. in 2 mltissue culture medium alone, or with 30 μg/ml LPS or with the indicatedphosphorothioate modified ODN at 1 μM. Cell cycle analysis was performedas described in (Darzynkiewicz, Z. et al., Proc. Natl. Acad. Sci. USA78:2881 (1981)).

To test the mitogenic effects of CpG ODN on human cells, perpheral bloodmonocyte cells (PBMCs) were obtained from two patients with chroniclymphocytic leukemia (CLL), a disease in which the circulating cells aremalignant B cells. Cells were cultured for 48 hrs and pulsed for 4 hourswith tritiated thymidine as described above.

Example 2 Effects of ODN on Production of IgM from B cells

Single cell suspensions from the spleens of freshly killed mice weretreated with anti-Thyl, anti-CD4, and anti-CD8 and complement by themethod of Leibson et al., J. Exp. Med. 154:1681 (1981)). Resting B cells(<02% T cell contamination) were isolated from the 63-70% band of adiscontinuous Percoll gradient by the procedure of DeFranco et al, J.Exp. Med. 155:1523 (1982). These were cultured as described above in 30μM ODN or 20 μg/ml LPS for 48 hr. The number of B cells activelysecreting IgM was maximal at this time point, as determined by ELIspotassay (Klinman D. M. et al. J. Immunol. 144:506 (1990)). In that assay,B cells were incubated for 6 hrs on anti-Ig coated microtiter plates.The Ig they produced (>99% IgM) was detected using phosphatase-labelledanti-Ig (Southern Biotechnology Associated, Birmingham, Ala.). Theantibodies produced by individual B cells were visualized by addition ofBCIP (Sigma Chemical Co., St. Louis Mo.) which forms an insoluble blueprecipitate in the presence of phosphatase. The dilution of cellsproducing 20-40 spots/well was used to determine the total number ofantibody-secreting B cells/sample. All assays were performed intriplicate. In some experiments, culture supernatants were assayed forIgM by ELISA, and showed similar increases in response to CpG-ODN.

table 1

Example 3 B Cell Stimulation by Bacterial DNA

DBA/2 B cells were cultured with no DNA or 50 μg/ml of a) Micrococcuslysodeikticus; b) NZB/N mouse spleen; and c) NFS/N mouse spleen genomicDNAs for 48 hours, then pulsed with ³H thymidine for 4 hours prior tocell harvest. Duplicate DNA samples were digested with DNAse I for 30minutes at 37 C prior to addition to cell cultures. E coli DNA alsoinduced an 8.8 fold increase in the number of IgM secreting B cells by48 hours using the ELISA-spot assay.

DBA/2 B cells were cultured with either no additive, 50 μg/ml LPS or theODN 1; 1a; 4; or 4a at 20 μM. Cells were cultured and harvested at 4, 8,24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10,20, 40 or 80 μM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wellswith no ODN had 3833 cpm. Each experiment was performed at least threetimes with similar results. Standard deviations of the triplicate wellswere <5%.

Example 4 Effects of ODN on Natural Killer (NK) Activity

10×10⁶ C57BL/6 spleen cells were cultured in two ml RPMI (supplementedas described for Example 1) with or without 40 μM CpG or non-CpG ODN forforty-eight hours. Cells were washed, and then used as effector cells ina short term ⁵¹Cr release assay with YAC-1 and 2C11, two NK sensitivetarget cell lines (Ballas, Z. K. et al. (1993) J. Immunol. 150:17).Effector cells were added at various concentrations to 10⁴ ⁵¹Cr-labeledtarget cells in V-bottom microtiter plates in 0.2 ml, and incubated in5% CO₂ for 4 hr. at 37° C. Plates were then centrifuged, and an aliquotof the supernatant counted for radioactivity. Percent specific lysis wasdetermined by calculating the ratio of the ⁵¹Cr released in the presenceof effector cells minus the ⁵¹Cr released when the target cells arecultured alone, over the total counts released after cell lysis in 2%acetic acid minus the ⁵¹Cr cpm released when the cells are culturedalone.

Example 5 In Vivo Studies with CpG Phosphorothioate ODN

Mice were weighed and injected IP with 0.25 ml of sterile PBS or theindicated phophorothioate ODN dissolved in PBS. Twenty four hours later,spleen cells were harvested, washed, and stained for flow cytometryusing phycoerythrin conjugated 6B2 to gate on B cells in conjunctionwith biotin conjugated anti Ly-6A/E or anti-Ia^(d) (Pharmingen, SanDiego, Calif.) or anti-Bla-1 (Hardy, R. R et al., J. Exp. Med. 159:1169(1984). Two mice were studied for each condition and analyzedindividually.

Example 6 Titration of Phosphorothioate ODN for B Cell Stimulation

B cells were cultured with phosphorothioate ODN with the sequence ofcontrol ODN 1a or the CpG ODN 1d and 3 Db and then either pulsed after20 hr with ³H uridine or after 44 hr with ³H thymidine before harvestingand determining cpm.

Example 7 Rescue of B Cells From Apoptosis

WEHI-231 cells (5×10⁴/well) were cultured for 1 hr. at 37 C in thepresence or absence of LPS or the control ODN 1a or the CpG ODN. 1d and3 Db before addition of anti-IgM (1 μ/ml). Cells were cultured for afurther 20 hr. before a 4 hr. pulse with 2 μCi/well ³H thymidine. Inthis experiment, cells with no ODN or anti-IgM gave 90.4×10³ by additionof anti-IgM. The phosphodiester ODN shown in Table 1 gave similarprotection, though with some nonspecific suppression due to ODNdegradation. Each experiment was repeated at least 3 times with similarresults.

Example 8 In Vivo Induction of IL-6

DBA/2 female mice (2 mos. old) were injected 1P with 500 μg CpG orcontrol phosphorothioate ODN. At various time points after injection,the mice were bled. Two mice were studied for each time point. IL-6 wasmeasured by Elisa, and IL-6 concentration was calculated by comparisonto a standard curve generated using recombinant IL-6. The sensitivity ofthe assay was 10 pg/ml. Levels were undetectable after 8 hr.

Example 9 Binding of B cell CREB/ATF to a Radiolabelled DoublestrandedCRE Probe (CREB).

Whole cell extracts from CH12.LX B cells showed 2 retarded bands whenanalyzed by EMSA with the CRE probe (free probe is off the bottom of thefigure). The CREB/ATF protein(s) binding to the CRE were competed by theindicated amount of cold CRE, and by single-stranded CpG ODN, but not bynon-CpG ODN.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-36. (Canceled).
 37. A method of increasing interferon-gamma in asubject comprising administering to a subject an immunostimulatoryoligonucleotide/delivery complex, said complex comprising animmunostimulatory oligonucleotide linked to a biodegradable deliverycomplex, wherein the immunostimulatory oligonucleotide comprises thesequence 5′-C, G-3′ and wherein said delivery complex is less than 10 μmin size, in an amount sufficient to modulate an immune response in saidsubject.
 38. The method of claim 37, wherein said delivery complex is aliquid phase microcarrier.
 39. The method of claim 37, wherein saidimmunostimulatory oligonucleotide is covalently linked to said deliverycomplex.
 40. The method of claim 37, wherein said immunostimulatoryoligonucleotide is non-covalently linked to said delivery complex. 41.The method of claim 37, wherein said immunostimulatory oligonucleotidecomprises a phosphate backbone modification.
 42. The method of claim 41,wherein said phosphate backbone modification is a phosphorothioate. 43.The method of claim 37, wherein the immunostimulatory oligonucleotide is8 to 40 nucleotides in length and comprises: 5′X₁X₂CGX₃X₄3′, wherein Cand G are unmethylated and X₁, X₂, X₃, and X₄ are nucleotides.
 44. Themethod of claim 43, wherein the immunostimulatory oligonucleotide doesnot include a GCG trinucleotide at a 5′ and/or 3′ terminal.
 45. Themethod of claim 43 wherein the immunostimulatory oligonucleotide doesnot contain a 5′X₁X₂CGX₃X₄3′ palindrome.
 46. The method of claim 44wherein the immunostimulatory oligonucleotide does not contain a5′X₁X₂CGX₃X₄3′ palindrome.